Journal: Sub-cellular biochemistry

Article Title: Targeting p53-MDM2-MDMX Loop for Cancer Therapy

doi: 10.1007/978-94-017-9211-0_16

Figure Lengend Snippet: Inhibitors targeting MDM2-MDMX loop discovered by different strategies

Article Snippet: Over the past decade, a number of new small molecules in addition to the previously reported Nutlin-3 [ 44 ] have been identified to target either MDM2 or MDMX or both in this pathway, some of which as listed in have been further advanced to the stage of clinical trials. table ft1 table-wrap mode="anchored" t5 caption a7 Small molecule Status ID Scaffold Discover methods K i or IC 50 In vivo test Reference M2-p53 interaction a Preclinical Nutlin-3 c Imidazolines HTS, SPR assay ~0.1 µM Retinoblastoma [ 44 ] Phase I RG7112 (RO5045337) 18 nM Advanced malignancies, except leukemia [ 100 ] M2-p53 interaction Preclinical MI63, MI-219 Spirooxindoles Structure-based design ~1–50 nM SJSA-1 xenograft [ 94 ] Phase I MI-773 (SAR405838) Advanced cancer [ 63 ] M2-p53 interaction NSC333003 c Benzothiazoles Virtual screening ~20 µM [ 150 ] M2-p53 interaction Preclinical TPD222669 Benzodiazepines HTS ThermoFlor 0.08 µM A3 7 5 xenograft [ 151 ] M2-p53 interaction Preclinical YH265 Pyrazoles HCS, Biosensor 1.8 µM NCI60 cancer cells [ 83 , 84 ] WO2011106650 M2-p53 interaction Phase I CGM097 (Novartis) <50 nM Selected advanced and refractory solid tumors [ 63 ] M2-p53 interaction Phase I MK-8242 (Merck) Piperidines [ 63 ] M2-p53 interaction Preclinical PXN727 (Priaxon) Isoquinoline-1-ones, pyrrolinones Virtual screening 8 nM-1 µM LNCaP xenograft http://www.priaxon.com/content/_docs/docl264519272209.pdf M2-p53 interaction Tetra-substituted heteroaryls Structure-based design 1 nM-50 µM [ 63 ] M2-p53 interaction c Substituted pyrazoles Structure-based design M2: ~0.02 µM; MX: ~10 µM M2-p53 interaction c Indole-2-carboxylic acids Structure-based design 130–400 nM M2-p53 interaction Indolinones Structure-based design 1.2nM-3.7nM M2-p53 interaction Piperidines Structure-based design 0.02–0.27 µM M2-p53 interaction Preclinical AM-8553 Piperidinones Structure-based design 150 nM SJSA xenograft [ 96 ] M2-p53 interaction Preclinical Pyrrolidone Virtual screening, FP assay 0.26 µM A549 xenograft [ 152 ] MX-p53 interaction b Preclinical SJ-172550 c SJ000558295 Imidazolines HTS,FP assay HTS.FP ~1 µM M2:9.1 µM; MX: ~9.0 µM Retinoblastoma cells [ 59 ] [ 63 ] Preclinical SJ000558304 HTS.FP M2:>150nM; MX: ~2 µM MX-p53 interaction XI-006 Benzofuroxans HCS, luciferase reporter assay MCF-7 cells [ 60 ] Dual inhibitor Preclinical RO-2443, RO-5963 c Indolyl Hydantoins HTS,TR-FRET Assay M2 and MX: <50 nM MCF7, HCT, RKO cells [ 61 ] Dual inhibitor?

Techniques: In Vivo, SPR Assay, FP Assay, Luciferase, Reporter Assay, Cell Based Assay

Journal: Communications Biology

Article Title: CRISPR activation screen in mice identifies novel membrane proteins enhancing pulmonary metastatic colonisation

doi: 10.1038/s42003-021-01912-w

Figure Lengend Snippet: a Graphic representation of the screen outline. b The number of metastatic colonies in the lungs of mice 10 days after being tail vein dosed with 2 × 10 5 B16-F0-dCas9 cells carrying gRNAs against the genes listed. NTC, non-targeting control. c The number of metastatic colonies in the lungs of mice 10 days after being tail vein dosed with 1 × 10 5 B16-F0 cells that have been stably transfected with a plasmid carrying the Lrrn4cl cDNA (LRN) or an empty vector (PB). d The number of metastatic colonies in the lungs of mice 10 days after being tail vein dosed with 4 × 10 5 B16-F0 cells that have been stably transfected with a vector carrying the Lrrn4cl cDNA (LRN) or an empty vector (PB). For (b-d), each symbol represents a mouse, the bars represent mean ± SD, 2 independent experiments performed (representative data from one experiment is shown) and statistics performed using a Mann–Whitney t test. e The human and mouse LRRN4CL protein sequences (ENSP00000325808 and ENSMUSP00000093976, respectively) were aligned CLUSTAL W (1.81) in Ensembl. Below each site amino acid of the alignment is a key denoting conserved sites (*), sites with conservative replacements (:), sites with semi-conservative replacements (.), and sites with non-conservative replacements (). The UniProtKB predicted location of the signal peptide (orange), extracellular domain (blue), transmembrane domain (red), cytoplasmic domain (green), FN3 domain (underlined) and N-linked glycosylation site at N132 (arrow) for human LRRN4CL are shown (glycosylation sites for the mouse also shown: arrows at N132 and N174).

Article Snippet: Primary antibodies were as follows: Epithelial–Mesenchymal Transition Antibody Sampler Kit (note: expression of Slug was not detected in A375 cells; 1:1000 dilution, Cell Signalling #9782), Matrix Remodelling Antibody Sampler Kit (1:1000 dilution, Cell Signalling #73959), StemLight TM Pluripotency Transcription Factor Antibody Kit (note: expression of Nanog and Oct-4A was not detected in A375 cells; 1:1000 dilution, Cell Signalling #9093), LRRN4CL antibody—middle region (1:1000 dilution, Aviva Systems Biology, #OAAB0898), β-Actin (8H10D10) mouse monoclonal Antibody (1:2000 dilution, Cell Signalling #3700), α-Tubulin Antibody (1:1000 dilution, Cell Signalling #2144), and mouse monoclonal anti-FLAG ® M2-Peroxidase (1:5000 dilution, Sigma #A8592).

Techniques: Control, Stable Transfection, Transfection, Plasmid Preparation, MANN-WHITNEY, Glycoproteomics

Journal: Communications Biology

Article Title: CRISPR activation screen in mice identifies novel membrane proteins enhancing pulmonary metastatic colonisation

doi: 10.1038/s42003-021-01912-w

Figure Lengend Snippet: a The number of metastatic colonies in the lungs of mice 10 days after being tail vein dosed with melanoma cell lines (5 × 10 5 HCmel12 cells, 4 × 10 5 YUMM1.7 cells and 0.25 × 10 5 B16-BL6 cells) that have been stably transfected with a vector carrying mouse Lrrn4cl cDNA (LRN) or an empty vector (PB). b The number of metastatic colonies in the lungs of mice 10 days after being tail vein dosed with a colorectal cell line (4 × 10 5 MC-38 cells), bladder cancer cell line (4 × 10 5 MB-49 cells), or breast cancer cell line (4 × 10 5 EO771 cells) that have been stably transfected with a vector carrying mouse Lrrn4cl cDNA (LRN) or an empty vector (PB). c The number of metastatic colonies in the lungs of mice 30 days after being tail vein dosed with human melanoma cell lines (2 × 10 5 A375 cells or 0.5 × 10 5 MeWo cells) or 10 days after being dosed with a mouse melanoma cell line (4 × 10 5 B16-F0 cells) that have been stably transfected a vector carrying human LRRN4CL cDNA (LRN) or an empty vector (PB). For a–c, each symbol represents a mouse, the bars represent mean ± SD, two independent experiments were performed (representative data from one experiment is shown) and statistics were performed using a Mann–Whitney t test. d The number of metastatic colonies in the livers of mice after being tail vein dosed (with 5 × 10 5 HCmel12 cells (wild-type mice) or 1 × 10 5 B16-F0 cells (NOD-SCID mice)) or intrasplenic dosed (0.1 × 10 5 B16-F0 cells (wildtype mice)) with melanoma cells that have been stably transfected a vector carrying mouse Lrrn4cl cDNA (LRN) or an empty vector (PB). Each symbol represents a mouse, the bars represent mean ± SD, and statistics were performed using a Mann–Whitney t test.

Article Snippet: Primary antibodies were as follows: Epithelial–Mesenchymal Transition Antibody Sampler Kit (note: expression of Slug was not detected in A375 cells; 1:1000 dilution, Cell Signalling #9782), Matrix Remodelling Antibody Sampler Kit (1:1000 dilution, Cell Signalling #73959), StemLight TM Pluripotency Transcription Factor Antibody Kit (note: expression of Nanog and Oct-4A was not detected in A375 cells; 1:1000 dilution, Cell Signalling #9093), LRRN4CL antibody—middle region (1:1000 dilution, Aviva Systems Biology, #OAAB0898), β-Actin (8H10D10) mouse monoclonal Antibody (1:2000 dilution, Cell Signalling #3700), α-Tubulin Antibody (1:1000 dilution, Cell Signalling #2144), and mouse monoclonal anti-FLAG ® M2-Peroxidase (1:5000 dilution, Sigma #A8592).

Techniques: Stable Transfection, Transfection, Plasmid Preparation, MANN-WHITNEY

Journal: Communications Biology

Article Title: CRISPR activation screen in mice identifies novel membrane proteins enhancing pulmonary metastatic colonisation

doi: 10.1038/s42003-021-01912-w

Figure Lengend Snippet: a The number of CFDA-positive B16-F0 cells present in the lung (per 2 million lung cells counted) after either 1- or 3-h post-tail vein dosing. The cells were stably transfected with a vector carrying mouse Lrrn4cl cDNA (LRN) or an empty vector (PB), and then labelled with the fluorescent tracker dye, CFDA. Each symbol represents a mouse, the bars represent mean ± SD, and statistics were performed using a Mann–Whitney t test. b The number of metastatic colonies in the lungs of either wildtype or immunodeficient (NOD-SCID) mice 10 days after being tail vein dosed with B16-F0 cells that have been stably transfected with a vector carrying mouse Lrrn4cl cDNA (LRN) or an empty vector (PB). Each symbol represents a mouse, the bars represent mean ± SD, two independent experiments were performed (representative data from one experiment is shown) and statistics were performed using a Mann–Whitney t test. c The growth of B16-F0 cells that have been stably transfected with a vector carrying mouse Lrrn4cl cDNA (LRN) or an empty vector (PB) after subcutaneous administration into the flank of wildtype mice. The data are shown as mean ± SEM with n = 8–9 mice per cell line per experiment. Two independent experiments were performed (representative data from one experiment is shown).

Article Snippet: Primary antibodies were as follows: Epithelial–Mesenchymal Transition Antibody Sampler Kit (note: expression of Slug was not detected in A375 cells; 1:1000 dilution, Cell Signalling #9782), Matrix Remodelling Antibody Sampler Kit (1:1000 dilution, Cell Signalling #73959), StemLight TM Pluripotency Transcription Factor Antibody Kit (note: expression of Nanog and Oct-4A was not detected in A375 cells; 1:1000 dilution, Cell Signalling #9093), LRRN4CL antibody—middle region (1:1000 dilution, Aviva Systems Biology, #OAAB0898), β-Actin (8H10D10) mouse monoclonal Antibody (1:2000 dilution, Cell Signalling #3700), α-Tubulin Antibody (1:1000 dilution, Cell Signalling #2144), and mouse monoclonal anti-FLAG ® M2-Peroxidase (1:5000 dilution, Sigma #A8592).

Techniques: Stable Transfection, Transfection, Plasmid Preparation, MANN-WHITNEY

Journal: Communications Biology

Article Title: CRISPR activation screen in mice identifies novel membrane proteins enhancing pulmonary metastatic colonisation

doi: 10.1038/s42003-021-01912-w

Figure Lengend Snippet: Cluster 6 and 7 of the heatmap of the differentially expressed genes (DEGs) between A375 melanoma cells over-expressing LRRN4CL (L) and A375 cells transduced with the empty vector (E) that were growing in vitro (VITRO) or in the lungs of mice for 21 days (VIVO). Supplementary Figure shows the full heatmap with all eight clusters. Three independent experiments were performed (cohorts 1–3) with four samples ( a – d ) per experiment per cell line (two samples are not shown on the heatmap as they were outliers on the PCA). Genes were scored as differentially expressed if they had a log 2 fold change ≤ −1 or ≥+1 with a Padj < 0.01. Individual genes within gene set enrichment analysis (GSEA)-identified significantly down-regulated pathways are shown (‘interferon_signaling’, ‘interferon_gamma’, ‘interferon_alpha’).

Article Snippet: Primary antibodies were as follows: Epithelial–Mesenchymal Transition Antibody Sampler Kit (note: expression of Slug was not detected in A375 cells; 1:1000 dilution, Cell Signalling #9782), Matrix Remodelling Antibody Sampler Kit (1:1000 dilution, Cell Signalling #73959), StemLight TM Pluripotency Transcription Factor Antibody Kit (note: expression of Nanog and Oct-4A was not detected in A375 cells; 1:1000 dilution, Cell Signalling #9093), LRRN4CL antibody—middle region (1:1000 dilution, Aviva Systems Biology, #OAAB0898), β-Actin (8H10D10) mouse monoclonal Antibody (1:2000 dilution, Cell Signalling #3700), α-Tubulin Antibody (1:1000 dilution, Cell Signalling #2144), and mouse monoclonal anti-FLAG ® M2-Peroxidase (1:5000 dilution, Sigma #A8592).

Techniques: Expressing, Transduction, Plasmid Preparation, In Vitro

Journal: Communications Biology

Article Title: CRISPR activation screen in mice identifies novel membrane proteins enhancing pulmonary metastatic colonisation

doi: 10.1038/s42003-021-01912-w

Figure Lengend Snippet: a LRRN4CL expression (RSEM) amongst different tumour types (data from TCGA). Bar represents the median, box represents the interquartile range. Whiskers represent 1.5 * IQR. Abbreviations: HCC hepatocellular carcinoma, THCA well-differentiated thyroid cancer, LEUK leukaemia, SEM seminoma, RNCCC renal non-clear cell carcinoma, BMCN mature B-cell neoplasms, TET thymic epithelial tumour, CHOL cholangiosarcoma, NSCLC non-small cell lung cancer, HNSCC head and neck squamous cell carcinoma, RCCC renal clear cell carcinoma, CSCC cervical squamous cell carcinoma, OSCC oesophageal squamous cell carcinoma, NCGCT non-seminomatous germ cell tumour, BRCA invasive breast carcinoma, GBMLGG diffuse glioma, UC bladder urothelial carcinoma, COADCA colorectal adenocarcinoma, CEAD cervical adenocarcinoma, PCC pheochromocytoma, OSCA oesophagogastric neuroepithelial tumour, MNET miscellaneous neuroepithelial tumour, OET ovarian epithelial tumour, ACC adrenocortical carcinoma, UCEC endometrial carcinoma, MESO pleural mesothelioma, PRAD prostate adenocarcinoma, PAAD pancreatic adenocarcinoma, GBM glioblastoma, SARC sarcoma, SKCM cutaneous melanoma, UM uveal melanoma. b Kaplan–Meier curves for disease-specific survival across all cancer patients from the TCGA dataset. Expression levels of LRRN4CL as lower 25th percentile (‘low LRRN4CL ’) or upper 25th percentile (‘high LRRN4CL ’). Cox Log-rank P value, age- and sex-adjusted. c Kaplan–Meier curves for disease-specific survival in cutaneous melanoma patients from the TCGA dataset. Expression levels of LRRN4CL as lower 25th percentile (‘low LRRN4CL ’) or upper 25th percentile (‘high LRRN4CL ’). Cox Log-rank P value, age- and sex-adjusted. d Kaplan–Meier curves for progression-free survival in cutaneous melanoma patients from the phase III adjuvant AVAST-M dataset . Expression levels of LRRN4CL as lower 25th percentile (‘low LRRN4CL ’) or upper 25th percentile (‘high LRRN4CL ’). Cox Log-rank P value, univariate analysis. The numbers in brackets on each graph indicate the number of patient samples per group.

Article Snippet: Primary antibodies were as follows: Epithelial–Mesenchymal Transition Antibody Sampler Kit (note: expression of Slug was not detected in A375 cells; 1:1000 dilution, Cell Signalling #9782), Matrix Remodelling Antibody Sampler Kit (1:1000 dilution, Cell Signalling #73959), StemLight TM Pluripotency Transcription Factor Antibody Kit (note: expression of Nanog and Oct-4A was not detected in A375 cells; 1:1000 dilution, Cell Signalling #9093), LRRN4CL antibody—middle region (1:1000 dilution, Aviva Systems Biology, #OAAB0898), β-Actin (8H10D10) mouse monoclonal Antibody (1:2000 dilution, Cell Signalling #3700), α-Tubulin Antibody (1:1000 dilution, Cell Signalling #2144), and mouse monoclonal anti-FLAG ® M2-Peroxidase (1:5000 dilution, Sigma #A8592).

Techniques: Expressing, Adjuvant